Method of gel chromatography



Oct. 27, 1970 A. J. FRISQUE ET AL 3,536,614

METHOD OF GEL CHROMATOGRAPHY 2 Sheets-Sheet 1 Filed June 9, 1969 I k. mvWk @(QDUWQ S 1 s r nM m w q M emwkzr LnF n .m; a wm D J M w 0% H m DLN M1? T m Oct. 27, 1970 A. J. FRISQUE ETA!- 3,536,614

METHOD OF GEL CHROMATQGRAPHY 2 Sheets-Sheet 2 Filed June 9, 1969 A RQM13J0 AWN .YN NN ON Q Q E N. 9

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Alvin J. Frisque Jerilgnn S.Re s m.

United States Patent 3,536,614 METHOD OF GEL CHROMATOGRAPHY Alvin J.l-h'isque, La Grange, and Jerilynn S. Resan, Chicago, Ill., assignors toNalco Chemical Company, Chicago, 11]., a corporation of Delaware FiledJune 9, 1969, Ser. No. 831,502 Int. Cl. B0ld 15/08 U.S. Cl. 210-31 4Claims ABSTRACT OF THE DISCLOSURE Polymer fractions in terms ofmolecular weight may be separated using a sieving gel which is a highlycrosslinked polymer based on water soluble adducts of methylenebisacrylamide.

This invention relates to highly water insoluble sieving gels and bedsmade therefrom capable of fractionating water soluble polymers withmolecular weights in the range of 10 40". The gel of the presentinvention is water compatible in that the discrete solid polymerparticles (each a substantially spherical bead or grain) internally trapwater.

A sieving gel bed is deemed herein to be a gel filter medium serving,when present as a distinct bed or mass in a container, to separate largemolecules from smaller molecules poured onto the gel filter bed. Thetechnology is known as gel chromatography.

Briefly, a polymeric system of diverse molecular weight, containing bothlarge and small molecules in solution, as to which separation bymolecular weight is to be performed, is presented to the sieving gelimmersed in an aqueous medium. The larger molecules, incapable ofpenetrating the gel beads, remain in solution and move through the outerspaces between the gel grains or beads, whereas the smaller moleculespenetrate the gel grains which themselves are porous and contain aninternal solvent which under the present invention is water or otherpolar liquid.

In other Words given a gel bed of a certain height, the larger moleculesin the ideal circumstance will be concentrated at the bottom, and thelighter ones tend to penetrate the gel grains at a higher level. Anotherpicture or model is that the larger molecules stay outside the gelgrains, in the outer solvent, whereas the smaller molecules are trappedin the interior solvent (water) inside the porous gel grains. Thepolymeric fractions may be collected by elution liquid displacement asdescribed in U.S. Pat. No. 3,369,007. The greater the porosity of thegel bed, the greater is the efficiency in separating the polymers ofgreater molecular weight.

The sieving gel of U.S. Pat. No. 3,369,007 is a copolymer of analkylidene-bisacrylamide monomer and an ethylenic monomer. We have foundthat a superior sieving gel, a more rigid or dense one, may be producedby polymerizing highly water soluble adducts of the bisacrylamide whichitself is only slightly water soluble. Methylene bisacrylamide, forexample, is water soluble only to the extent of 3% by weight C.) whereasthe monomers used for emulsion polymerization under the presentinvention are at least ten times more soluble, and hence there can be100% cross-linker in the course of forming the polymers, compatiblysurrounding Water droplets, to complete the discrete, spherical gelgrains or beads.

Thus the sieving gel of the present invention is characterized by rigidgrains, more so than the product of U.S. Pat. No. 3,369,007, which is tosay that the polymeric beads or grains of the present invention are lesselastic. Resultantly, the gel bed is less compacted, or in 3,536,614Patented Oct. 27, 1970 "ice other words greater macroporosity isachieved characterizing high fiowability or permeability, and thereforeincreased efficiency in fractionating higher molecular weight polymerscompared to the gel bed in U.S. Pat. No. 3,369,007.

It is therefore the primary object of the present invention to produce awater-compatible sieving gel of sufficient macroporosity to separatepolymers in the molecular weight range of l0 l0 not heretofore achievedwith such gels, and an object related thereto is to attain a high degreeof macroporosity by producing beads or grains that are relatively rigidin nature. Another object of the present invention is to achieve suchrigid structures by producing the gel polymer from highly water solublemonomers capable of developing a high degree of crosslinking.

In the drawings:

FIG. 1 is a graph on logarithmic scale showing the separation of polymerfractions attained under the present invention, using a homopolymer gel;and

FIG. 2 is a graph on logarithmic scale showing the separation of polymerfractions obtained under the present invention, using a copolymer gel.

The structural formula for the water soluble adducts of the presentinvention may be written as:

where: R is selected from the group consisting of H, a C -C alkanegroup, COOH, CH CH O, and

CH CH CH O R and R are selected from the group consisting of CH OH,COOH, and HSO and R and R are selected from the group consisting of Hand CH Preferably:

R=H R,=COOH R2=H R3:H 4=

One method of attaining a water soluble adduct is to react methylenebisacrylamide with either formaldehyde or formic acid, yielding:

(formaldehyde adduct: 40 parts soluble per 100 parts solvent) HrHCOO- oN-iicrr=CH: CH2 I IO-HG=GII= mulcoo- (acid adduct: parts soluble perparts solvent).

Either adduct may be polymerized by itself (100% cross-linker") or itmay be copolynierized with an ethylenic (mono functional vinyl) compoundhaving the formula RCH=CH such as acrylamide, provided the adductmonomer is at a higher level than would be soluble as the non-adductedform, that is, at the same temperature as previously stated, this wouldbe more than 3 grams per 100 ml. solvent.

In producing the formaldehyde adduct, it is only necessary to react thebisacrylamide and formaldehyde on a mole-to-mole ratio in a basicsolution while heating. The acid adduct may be formed by reacting thebisacrylamide with the formic acid or formic acid and water.

EXAMPLE 1 Methylene bisacrylamide, 160 grams, is dissolved in 240 ml. offormic acid in the presence of 20 ml. of an oleate emulsifier such asTween 80, producing the acid adduct.

A catalyst solution is prepared consisting of 20 grams of (NI-[ 8 0dissolved in 40 ml. of water.

A reaction vessel containing 1500 ml. of heptane and 80 ml. of Span 80is purged with N for 30 minutes, stirring constantly at a speedsutlicient to disperse the aqueous phase as small drops within theorganic phase. The monomer and catalyst solutions are then added in a :1ratio. A homopolymer of the adduct is formed, crosslinking taking placebetween adjacent vinyl gloups.

The resultant gel is cured at a temperature of 50 C.

for about two hours. After cooling, the product is poured through abasket centrifuge and washed extensively with alcohol followed by awater wash. Size distribution is selected by wet sieving.

EXAMPLE 2 Methylenebisacrylamide, 90 grams, is dissolved in 240 ml. offormaldehyde solution at 50 C. and a pH of 8-10 in the presence of ml.of an oleate emulsifier such as Tween 80, producing the formaldehydeadduct.

A catalyst solution is prepared consisting of 20 grams of (NH S Odissolved in 40 1111. of water.

A reaction vessel containing 1500 ml. of heptane and 80 ml. of Span 80is purged with N for minutes, stirring constantly at a speed sufficientto disperse the aqueous phase as small drops within the organic phase.The monomer and catalyst solutions are then added in a 5:1 ratio. Ahomopolymer of the adduct is formed, crosslinking taking place betweenadjacent vinyl groups.

The percent gel is expressed as grams/100 ml. of solvent, which is theinner water content of the gel beads in this instance. Gels made fromthe formaldehyde adduct range from 3% to from the acid adduct, 3% to80%; for an adduct and acrylamide (copolymer), 3% to 150%. Mesh sizesrange from to 400, controlled by commonly known emulsion technology.

FIG. 1 presents laboratory gel chromatography data showing attainment ofmolecular sieving in the range of 10 -10 using gels of different percentbut made entirely from the acid adduct of Example 1, 100% cross-linker.

The lower curve, FIG. 1, shows that a gel made by polymerizing grams ofthe water soluble bisacrylamide (bis) adduct per ml. solvent (80%)fractionates polymers between 10 and 10 molecular weight (M.W.); theupper curve, that a 6% gel made by polymerizing 6 grams of "bis" per 100ml. solvent fractionates polymers between 10 and 10 M.W.

The abscissa of FIG. 1 represents the fraction of the internal volume ofthe gel particle available to the molecular weight shown. For example,with the 80% gel 0.7 of the internal volume is available to M.W. 300 and0.1 is available to M.W. 10,000; for the 6% gel, 0.9 of the internalvolume is available to M.W. about 10,000, and only about 0.1 isavailable to M.W. 5,000.000. Water compatible acrylamide gelscommercially available exclude polymers of 300,000 M.W., which are theaPP mate exclusion limits of a 25% gel of the present invention.

Among the commercial water compatible sieving gels, agarose, which hasthe highest permeability limit, excludes a substantial fraction of 2 10M.W. polymers, even at a solids content (gel) as low as 2%. On the otherhand, the present 6% gel, FIG. 1, completely includes this fractron.

FIG. 2 shows molecular separations using a copolymer of acrylamide andthe formaldehyde adduct of bis, there being three dillerent ratios oftotal monomer content: bis" adduct monomer, namely, 40:50, 30:50 and20:50 per 100 ml. of solvent (water). It may be mentioned that indesignating a 40:50 ratio, for example, this means that per 100 ml.polar solvent there are 40 grams of gel (40%) of which 50% is the bisadduct, and 50% is acrylamide. The lowest acceptable limit appears to bemore than 3% by weight of the bis" adduct in the gel polymer, and it maybe 100%.

It will be seen from the foregoing that under the present invention weafford a water compatible sieving gel capable of separating fractions ofmolecular weight nearly 10 and we accomplish this by having resort togel polymers which include a relatively high amount of cross-linker inthe form of a water soluble adduct of methylene bisacrylamide.

Hence while we have disclosed preferred embodiments of our invention itis to be understood that these are capable of variation andmodification.

We claim:

1. A water compatible sieving gel selected from the group consisting of(1) a homopolymer of a water soluble bisacrylamide monomer having theformula li Th u; in

where: R is selected from the group consisting of H, a C -C alltanegroup, -COOH, CH CH O-, and

R and R are selected from the group consisting of CH OH, -COOH. and HSOand R and R are selected from the group consisting of H and CH and (2) acopolymer of said bisacrylamide and a vinyl monomer standing in theratio of greater than 3:97

grams per 100 ml. polar solvent.

2. A gel according to claim 1 in which R, R R and R are H, R is CH OH,and the vinyl monomer is acrylamide.

3. A gel according to claim 1 in which R, R R arid R are H, R is -COOHand the vinyl monomer is acrylamide.

4. A method of gel chromatography in which the gel bed is represented bygel particles selected from the group consisting of (1) a homopolymer ofa Water soluble bisacrylamide monomer having the formula ll--C C:CII211("211 ITTM-YICH2 R3 0 1h where: R is selected from the groupconsisting of H, A(

alkane group, -COOH, CH CH O, and

CH CH CH O- R and R are selected from the group consisting of Referen esCited CH2OH, "COOH, and 41504; and UNITED STATES PATENTS R fiaggdRc Hageselected /from the group consistmg of 3,298,925 1/1967 Mosbach u 2 1031X J. L. DECESARE, Primary Examiner and (2) a copolymer of saidbisacrylamide and a vinyl Us. CL X'R.

monomer standing in the ratio of greater than 3:97 grams per 100 ml.polar solvent.

